Botulinum neurotoxin (BoNT) is the most toxic substance known to science. Recent advances in molecularly engineered vaccines have shown that multiple doses of recombinant C-terminal domains (Hc) of BoNT confer protection in animals challenged with 10(6) LD50. The goal of this Project is to develop a more efficient vaccination schedule: live vector-based multivalent botulinum vaccines will be developed to prime vaccinees such that they will mount responses that are faster, higher and stronger than without such priming. We will develop 2 "generations" of S. Typhi-based live vector vaccines. The first generation (Category 1) consists of live vectors expressing individual Hc domains from 4 serotypes; the second generation (Category 2), will yield single live vector strains expressing up to 4 different Hc serotypes. It is our intention to co-administer the final mutivalent product with a cholera-based vaccine (Project 4) against the remaining 3 serotypes. Products 1 - 4: The attenuated S. Typhi vaccine CVD 908-htrA will be used as a live vector to individually express BoNT/A-Hc, BoNT/B-Hc BoNT/E-Hc. and BoNT/F-Hc from a genetically stabilized expression plasmid. The individual Hc serotypes will be targeted to the periplasmic space, the bacterial cell surface, or will be exported out of CVD 908-htrA into the extracellular environment. Products 5 - 7: Using the constructs and information generated in development of Products 1- 4, we will proceed to co-express subcellular location-optimized BoNT/A-Hc, B-Hc and F-Hc simultaneously from a single operon, encoded by a genetically stabilized expression plasmid in CVD 908-htrA (Product 5). Related to Product 5, Product 6 will co-express subcellular location-optimized BoNT/B-Hc, E-Hc, and F-Hc from a single operon encoded by a genetically stabilized expression plasmid. Product 7 will comprise CVD 908-htrA in which the four Hc serotypes are encoded by a single operon contained on a stabilized expression plasmid, on which transcriptional control of the operon has been optimized. These remaining 3 products will be ready for human testing by the end of the fifth year of this proposal. We expect that one or more of the second generation vaccines should result in excellent priming of the human immune system.